Glucose measurement utilizing non-invasive assessment methods

ABSTRACT

This involves non-invasive glucose measurement processes for determining blood glucose level in the human body. After achieving a static level of glucose at a surface of the skin over some period of time, the glucose may then be measured by a variety of different processes. A sample of the glucose may also first be extracted from the skin and this sample may then be measured. Clearly, these processes are especially suitable for monitoring glucose levels in the human body, and is especially beneficial to users having diabetes mellitus. These procedures may be used for other analyte materials that are found in appropriate regions of the outer skin.

FIELD OF THE INVENTION

This invention involves non-invasive glucose measurement and a processfor determining blood glucose levels in the human body. Preferably, theprocess is used on a fingertip or other part of the body, typically askin surface of the body.

BACKGROUND OF THE INVENTION

The American Diabetes Association reports that nearly 6% of thepopulation in the United States, a group of 16 million people, hasdiabetes. The Association further reports that diabetes is the seventhleading cause of death in the United States, contributing to nearly200,000 deaths per year. Diabetes is a chronic disease having no cure.The complications of the disease include blindness, kidney disease,nerve disease, and heart disease, perhaps with stroke. Diabetes is saidto be the leading cause of new cases of blindness in individuals in therange of ages between 20 and 74; from 12,000-24,000 people per year losetheir sight because of diabetes. Diabetes is the leading cause ofend-stage renal disease, accounting for nearly 40% of new cases. Nearly60-70% of people with diabetes have mild to severe forms of diabeticnerve damage which, in severe forms, can lead to lower limb amputations.People with diabetes are 2-4 times more likely to have heart disease andto suffer strokes.

Diabetes is a disease in which the body does not produce or properly useinsulin, a hormone needed to convert sugar, starches, and the like intoenergy. Although the cause of diabetes is not completely understood,genetics, environmental factors, and viral causes have been partiallyidentified.

There are two major types of diabetes: Type I and Type II. Type Idiabetes (formerly known as juvenile diabetes) is an autoimmune diseasein which the body does not produce any insulin and most often occurs inyoung adults and children. People with Type I diabetes must take dailyinsulin injections to stay alive.

Type II diabetes is a metabolic disorder resulting from the body'sinability to make enough, or properly to use, insulin. Type II diabetesaccounts for 90-95% of diabetes. In the United States, Type II diabetesis nearing epidemic proportions, principally due to an increased numberof older Americans and a greater prevalence of obesity and a sedentarylifestyle.

Insulin, in simple terms, is the hormone that unlocks the cells of thebody, allowing glucose to enter those cells and feed them. Since, indiabetics, glucose cannot enter the cells, the glucose builds up in theblood and the body's cells literally starve to death.

Diabetics having Type I diabetes typically are required toself-administer insulin using, e.g., a syringe or a pin with needle andcartridge. Continuous subcutaneous insulin infusion via implanted pumpsis also available. Insulin itself is typically obtained from porkpancreas or is made chemically identical to human insulin by recombinantDNA technology or by chemical modification of pork insulin. Althoughthere are a variety of different insulins for rapid-, short-,intermediate-, and long-acting forms that may be used variously,separately or mixed in the same syringe, use of insulin for treatment ofdiabetes is not to be ignored.

It is highly recommended by the medical profession that insulin-usingpatients practice self-monitoring of blood glucose (SMBG). Based uponthe level of glucose in the blood, individuals may make insulin dosageadjustments before injection. Adjustments are necessary since bloodglucose levels vary day to day for a variety of reasons, e.g., exercise,stress, rates of food absorption, types of food, hormonal changes(pregnancy, puberty, etc.) and the like. Despite the importance of SMBG,several studies have found that the proportion of individuals whoself-monitor at least once a day significantly declines with age. Thisdecrease is likely due simply to the fact that the typical, most widelyused, method of SMBG involves obtaining blood from a finger stick. Manypatients consider obtaining blood to be significantly more painful thanthe self-administration of insulin.

There is a desire for a less invasive method of glucose measurement.Methods exist or are being developed for a minimally invasive glucosemonitoring, which use body fluids other than blood (e.g., sweat orsaliva), subcutaneous tissue, or blood measured less invasively. Sweatand saliva are relatively easy to obtain, but their glucoseconcentration appears to lag in time significantly behind that of bloodglucose. Measures to increase sweating have been developed and seem toincrease the timeliness of the sweat glucose measurement, however.

Subcutaneous glucose measurements seem to lag only a few minutes behinddirectly measured blood glucose and may actually be a better measurementof the critical values of glucose concentrations in the brain, muscle,and in other tissue. Glucose may be measured by non-invasive orminimally-invasive techniques, such as those making the skin or mucousmembranes permeable to glucose or those placing a reporter molecule inthe subcutaneous tissue. Needle-type sensors have been improved inaccuracy, size, and stability and may be placed in the subcutaneoustissue or peripheral veins to monitor blood glucose with smallinstruments. See, “An Overview of Minimally Invasive Technologies”,Clin. Chem. September 1992; 38(9):1596-1600.

Truly simple, non-invasive methods of measuring glucose are notcommercially available.

SUMMARY OF THE INVENTION

This invention involves non-invasive glucose measurement and a processfor determining blood glucose levels in the human body upon achieving astatic level of glucose at a skin surface over a period of time.

Processes which are able to assess glucose concentrations predictablyfrom a skin surface may include a step of extracting a sample from theskin and then measuring that sample from the skin. Such sampleextraction processes may include suction blister extraction, wickextraction, microdialysis extraction, iontophoretic extraction,sontophoretic extraction, and chemically enhanced extraction. Aside fromthe extraction processes, non-invasive measurement processes may includeelectrochemical sensors (e.g., glucose electrodes), optochemical sensors(e.g., colorimetric strips), near-infrared spectroscopy (NIR),mid-infrared spectroscopy (MIR), infrared spectroscopy (IR), Ramanspectroscopy, photoacoustic spectroscopy, measurement of refractiveindex or scatter changes, fluorescent spectroscopy, and polarizationspectroscopy.

The processes for extraction and measurement are illustrative and arenot meant to be an exclusive list.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph representing a glucose level rising sharply andreaching a static value.

FIG. 2 shows a graph correlating glucose levels measured using aspecific variation of the device with glucose levels in the blooddetermined using a commercial device.

FIG. 3 shows a pair of glucose IR curves (taken before and after eating)for an individual having diabetes made using the inventive glucosemeasuring device.

FIG. 4 shows a graph comparing glucose levels in a non-diabeticindividual (taken before and after eating) made using the inventiveglucose measuring device and direct blood measurement. This graph showsthat the inventive procedure tracks blood glucose levels with minimumtime lag.

DESCRIPTION OF THE INVENTION

In non-invasively measuring analyte levels using mid-infrared (“MIR”),particularly using a device and methods as described in U.S. patentapplication Ser. No. 09/547,433, filed Apr. 12, 2000, entitled “INFRAREDATR GLUCOSE MEASUREMENT SYSTEM (II)”, which is herein incorporated byreference in its entirety, penetration of skin by IR ranged in only afew micrometers. Thus, because of this small penetration depth, thedescribed device may have been measuring glucose from the mixture ofoils and sweat that is pumped to the skin surface. Therefore, multiplealternative processes may be utilized in non-invasively measuringanalytes, particularly glucose, from a skin surface.

The device in co-pending application Ser. No. 09/547,433 described theuse of IR attenuated total reflectance (“ATR”) spectroscopy to detectand ultimately to determine the level of a selected analyte, preferablyblood glucose, in the human body. Preferably, the inventive device usedan ATR procedure in which the size and configuration of the crystalpermits a number of internal reflections before the beam is allowed toexit the crystal with its measured information.

We have found that the mid-IR spectrum does not penetrate into the skinto an appreciable level. Specifically, the skin is made up of a numberof layers: the outermost—the stratum corneum—is a layer substantiallyfree of interference from cholesterol, water, gamma globulin, albumin,and blood. It is a shallow outer region covering the stratum granulosum,the stratum spinosum, and the basal layer. The area between the basallayer to the outside is not vascularized. It is unlikely that any layerother than the stratum corneum is traversed by the mid-IR light involvedin this inventive device. Although we do not wish to be bound by theory,it is likely that the eccrine or sweat glands transport the glucose tothe outer skin layers for measurement and analysis by our inventions.

We have surprisingly found that a glucose measuring device madeaccording to the invention in application Ser. No. 09/547,433 is quiteeffective on the human skin of the hands and fingers. We have found thatthe glucose concentration as measured by the inventive devicescorrelates very closely with the glucose concentration determined by adirect determination from a blood sample. This is surprising in that theIR beam likely passes into the skin, i.e., the stratum corneum, for onlya few microns. It is unlikely in a fingertip that any blood is crossedby that light path. As discussed above, the stratum corneum is the outerlayer of skin and is substantially unvascularized. The stratum corneumis the final outer product of epidermal differentiation orkeratinization. It is made up of a number of closely packed layers offlattened polyhedral corneocytes (also known as squames). These cellsoverlap and interlock with neighboring cells by ridges and grooves. Inthe thin skin of the human body, this layer may be only a few cellsdeep, but in thicker skin, such as may be found on the toes and feet, itmay be more than 50 cells deep. The plasma membrane of the corneocyteappears thickened compared with that of keratinocytes in the lowerlayers of the skin, but this apparent deposition of a dense marginalband formed by stabilization of a soluble precursor, involucrin, justbelow the stratum corneum.

It is sometimes necessary to clean the skin exterior prior beforesampling to remove extraneous glucose from the skin surface. At leastwhen using IR spectra to measure glucose, it is important to selectcleaning materials having IR spectra that do not interfere with the IRspectra of glucose. We consider a kit of the following to be suitablefor preparation of the sample skin for the testing. The components are:a.) a glucose solvent, e.g., water or other highly polar solvent; b.) asolvent for removing the water, e.g., isopropanol, and c.) a skinsoftener or pliability enhancer not having significant IR peaks in thenoted IR regions, e.g., mineral oils such as those sold as “Nujol”.Preferably the b.) and c.) components are admixed, although they neednot be. Certain mixtures of the first two components may be acceptable,but only if the sampling situation is such that the solvents evaporatewithout IR spectrographically significant residue. We have also foundthat soap and its residue are sometimes a problem. Consequently,addition of a weak acid again not having significant IR peaks in thenoted IR regions, to the a.) component, i.e., the solvent for removingglucose, is desirable. The preferred weak acid is boric acid. Theinventive kit preferably is made up of sealed packets of the components,most preferably each packet containing an absorbent pad.

Method of Use

As noted in application Ser. No. 09/547,433, for IR measurement, it isdesirable both to clean the plate before use and to clean the exteriorsurface of the skin to be sampled. Again, we have found that theexterior skin is highly loaded with glucose that is easily removedpreferably by using the skin preparation kit, or, less preferably, bywashing the skin. Reproducible and accurate glucose measurements maythen be had in a period as short as one to ten minutes, generally lessthan five minutes, after cleaning the area of the skin to be measured.

We also note that, depending upon the design of a specific variation ofa device made according to the invention, periodic at least an initialcalibration of the device, using typical blood sample glucosedeterminations, may be necessary or desirable.

Alternate Methods of Use

We have also observed the following phenomena. In attempting to measureglucose using the mid IR apparatus described above, we noted that byusing a solvent on the surface which dissolved glucose, our proceduresshowed that the glucose level at the skin was substantially lowered, ifnot eliminated. However, as is shown in the examples, shortlythereafter, the glucose level began to rise sharply and consequentlywould reach static value which was correlateable in a consistent fashionto a glucose level found in the blood.

As shown in FIG. 1, the curve representing the glucose level risessharply and eventually plateaus over a time period, T. This glucoselevel may be measured by any of the devices and processes as discussedin application Ser. No. 09/547,433 or herein at predetermined timeperiods, ΔT, until the glucose level reaches a substantially staticvalue, thus representing the glucose level in the blood. As we havenoted above, the outer layer of the skin is not vascularized and thephysiological source of the glucose transport to the skin surface is notall together clear. Nevertheless, it is easily assessable and quiterepeatable. Our method of using mid IR to measure this glucose level isbelieved, on basic principals, only to penetrate the skin at best a fewmicrons. Other procedures which are non-invasive and which are able toassess glucose concentration upon this achievement of glucose stasislevel are similarly and predictably able to assess the glucose level inthe human body.

Such alternative procedures may provide for extracting a sample from theskin and then measuring the analyte, or glucose, level in the sample.Potential extraction methods may include blister suction and wickextraction, microdialysis extraction, iontophoretic extraction,sontophoretic extraction, and chemically enhanced extraction.

Sample measurement methods may include electrochemical sensors (e.g.,glucose electrodes), optochemical sensors (e.g., colorimetric strips),near-infrared spectroscopy (NIR), infrared spectroscopy (IR), Ramanspectroscopy, photoacoustic spectroscopy, measurement of refractiveindex or scatter changes, fluorescent spectroscopy, and polarizationspectroscopy. These methods of sample extraction and measurement areprovided.

Glucose Extraction

Blister suction and wick extraction are some of the most common methodsfor sampling subcutaneous interstitial tissue fluid, although blisterextraction is less invasive than the wick extraction technique.Microdialysis extraction involves calculating the concentrations ofcompounds, including skin glucose concentrations, which are in theextracellular water space. Microdialysis has been applied to peripheraltissue types, e.g., skin, muscle, adipose, eye, lung, liver, and bloodas well as having microdialysis probes implanted subcutaneously andperfused by a portable microinfusion pump. Finally, iontophoreticextraction involves noninvasive glucose measurement from subcutaneoustissue.

Glucose Measurement

Electrochemical Sensors

Electrochemical sensors (e.g., glucose electrodes) utilize electricalsignal as a direct consequence of some (chemical) process occurring at atransducer/analyte interface. Some implantable glucose sensors mayinclude electrocatalytic sensors, which are based on directelectro-oxidation of glucose on noble metal electrodes, and biosensors,which combine glucose-specific enzymes with electrochemical electrodes.

Such sensors may be fabricated by combining biologically activecomponents (e.g., enzymes, antibodies, cells, tissues or microorganisms)with some physical transducer. Biosensors may be direct enzymebiosensors or affinity sensors based on enzyme labeled immunoassays.Enzymes may be used as a molecular recognition element in glucosesensing while immunoassays may provide the ability to sense extremelylow amounts of an ahalyte. Electrochemical sensors may also includepiezoelectric, thermoelectric, and acoustic sensors used for glucosemeasurement by utilizing an enzyme-catalysed reaction to create ameasurable change in a physical parameter detectable by a transducer.

Optochemical Sensors

Optochemical sensors (e.g., colorimetric strips) are based on changes insome optical parameter due to enzyme reactions or antibody-antigenbonding at a transducer interface. Such sensors may include enzymeoptrodes and optical immunosensors and may also include differentmonitoring processes such as densitometric, refractometric orcalorimetric devices.

Electrochemical biosensors may be constructed on the amperometricprinciple which is based on the oxidation or reduction ofelectrochemically active substances. Such sensor may also be constructedto measure the changes in local pH due to the gluconic acid produced ata potentiometric sensor, usually a coated wire pH-selective electrode oran ion selective field effect transistor (ISFET). Also, electricalresistance changes during the overall process may be used as a basis forconductometric biosensors.

Moreover, potentiometric glucose sensors (e.g., coated wire sensors) maypotentially be utilized for implantable use. Coated wire sensors aregeneral easy to fabricate and are suitable for miniaturization todiameters of 50-200 μm. They may also be used in combination with astandard cardiographic (EKG) reference electrode.

Near-Infrared Spectroscopy (NIR)

The NIR region of the spectrum extends from 700 to 2500 nm (14,000-4000cm⁻). In this region, absorption bands are due to overtone vibrations ofanharmonic fundamental absorption bands to combinations of fundamentalabsorption bands primarily associated with C—H; O—H, and N—H stretchingvibrations. For overtone vibrations, only the first, second, and thirdovertones are usually seen with the magnitude of the absorption peakdiminishing substantially with overtone order. The NIR region may beattractive for quantitative spectroscopy since NIR instrumentation isreadily available.

In measuring aqueous glucose, the NIR region which lies between 2.0 and2.5 μm may be utilized. This region contains a relative minimum in thewater absorption spectrum and has readily identifiable glucose peakinformation. However, NIR spectra may generally be sensitive to a hostof factors including temperature, pH, and scattering.

Raman Spectroscopy

Raman spectra are typically observed when incident light at a frequencyv₀=c/λ₀ is inelastically scattered at frequencies v₀±v_(i). The loss(Stokes shift) or gain (anti-Stokes shift) of photon energy, and hencefrequency, is due to transitions of the rotational and vibrationalenergy states within the scattering molecule. Since the Raman spectrumis independent of excitation frequency, an excitation frequency may bechosen which is appropriate for a particular sample. However, a drawbackmay be that scatter and reabsorption in biological tissues may makedetection of Raman shifts due to physiological concentrations difficult.

Photoacoustic Laser Spectroscopy

Photoacoustic laser spectroscopy has been utilized for measuring glucoseconcentrations of human whole blood samples using pulsed laserphotoacoustic spectroscopy. Such a process may use, e.g., a CO₂ laseroperating with μJ pulse energy, to measure tiny changes of theabsorption coefficient of the sample caused by the variations of bloodglucose concentrations.

Refractive Index or Scatter Changes

Measurement of refractive index or scatter changes may be feasible tomeasure blood glucose by measuring the scattering coefficient of humanskin, e.g., by using optical sensors attached to the skin. Suchtechniques may be based on the fact that the refractive index of sugarsolution changes with the concentration of sugar.

Fluorescent Spectroscopy

There may be two categories for fluorescent spectroscopy:glucose-oxidase based sensors and affinity-binding sensors. Sensors inthe first category may use the electroenzymatic oxidation of glucose byglucose-oxidase (GOX) in order to generate an optically detectableglucose-dependent signal. The oxidation of glucose and oxygen formsgluconolactone and hydrogen peroxide.

Several methods for optically detecting the products of this reaction,and hence the concentration of glucose driving the reaction, may beutilized. Since oxygen is consumed in this reaction at a rate dependenton the local concentration of glucose, a fluorophore which is sensitiveto local oxygen concentration can also be used to quantify glucoseconcentration.

A method GOX based fluorescent sensor involves the redox mediatortetrathiafulvalene (TTF) whose oxidized form TTF⁺ reacts with thereduced form of GOX to reversibly form TTF⁰. Since TTF⁺ is absorbed inthe 540-580 nm range, a method for quantifying the presence of TTF⁺ (andhence glucose driving the production of reduced GOX) is available.

Another method involves the hydrogen peroxide (H₂O₂) generated from theGOX reaction with glucose reacting with bis(2,4,6-trichlorophenyl)oxalate (TCPO) to form a peroxyoxylate. Here, the peroxyoxylate formedtransfers chemiluminescent energy to an accepting fluorophore which inturn emits photons at a characteristic wavelength. The emission by thefluorophore is proportional to the glucose concentration and may bedetected optically.

Polarization Spectroscopy

Polarimetric quantification of glucose may be based on the principle ofoptical rotary dispersion (ORD) where a chiral molecule in an aqueoussolution rotates the plane of linearly polarized light passing throughthe solution. This rotation is due to a difference in the indices ofrefraction n_(L) and n_(R) for left- and right-circularly polarizedlight passing through a solution containing the molecule. Because themolecule has a chirality (or “handedness”), the angle of rotationdepends linearly on the concentration of the chiral species, the pathlength through the sample, and a constant for the molecule called thespecific rotation. Glucose in the body is dextrorotatory, i.e., rotateslight in a right-handed direction, and has a specific rotation of +52.6°dm⁻¹ (g/L)⁻¹.

EXAMPLES OF MID-IR USE Example 1

Using a commercially available IR spectrometer (Nicolet 510) having aZnSe crystal ATR plate (55 mm long, 10 mm wide, and 4 mm thick) wetested the inventive procedure. We calibrated the output of thespectrometer by comparing the IR signal to the values actually measuredusing one of the inventor's blood samples. The inventor used a bloodstick known as “Whisper Soft” by Amira Medical Co. and “GlucometerElite” blood glucose test strips sold by Bayer Corp. of Elkhart, Ind. Oneach of the various test days, the inventor took several test sticks andmeasured the glucose value of the resulting blood; the IR test was madeat the same approximate time.

As shown in the calibration curve of FIG. 2, the data are quiteconsistent. So, where the blood glucose concentration “B” is in (mg/dl)and “S” is the difference between the absorbance at the referencingregion and the measuring region as measured by the spectrometer:

B=[(1950)•S]−(17).

Example 2

In accordance with a clinical protocol, a diabetic was then tested.Curve 1 in FIG. 3 shows the IR absorbance spectrum of the test subject'sfinger before eating (and after fasting overnight) and curve 2 shows IRabsorbance spectrum of the same individual after having eaten.Incidentally, insulin was administered shortly after the measurement ofcurve 2.

In any event, the significant difference in the two peak heights at the9.75 micrometer wavelength and the equality of the two IR absorbancevalues at the 8.50 micrometer value shows the effectiveness of theprocedure in measuring glucose level.

Example 3

That the inventive glucose monitoring device non-invasively determinesblood glucose level and quickly follows changes in that blood glucoselevel is shown in FIG. 4. Using both the inventive procedure and acommercial glucose device, one of the inventors followed his glucoselevel for a single day. The blood sticks are considered to be accuratewithin 15% of the actual reading.

The results are shown in FIG. 4. Of particular interest is themeasurement just before 4:40 pm wherein the two values are essentiallythe same. A high sugar candy bar was eaten at about 4:45pm andmeasurements of glucose level were taken using the inventive procedureat about 5:03, 5:18, 5:35 and 5:50. A blood sample was taken at 5:35 andreflected almost the same value as that measured using the inventiveprocedure. Consequently, the procedure tracks that measured by the bloodvery quickly.

This invention has been described and specific examples of the inventionhave been portrayed. The use of those specifics is not intended to limitthe invention in any way. Additionally, to the extent there arevariations of the invention with are within the spirit of the disclosureand yet are equivalent to the inventions found in the claims, it is ourintent that this patent will cover those variations as well.

We claim as our invention:
 1. A process for determining a glucose levelof a human body, comprising the steps of: a.) cleaning a skin surface onsaid human body; b.) achieving a non-induced static level of glucose atthe skin surface as measured over a period of time, wherein the staticlevel is correlateable to the glucose level of the human body; and c.)measuring said skin surface glucose level.
 2. The process of claim 1,wherein the step of measuring further comprises: a.) extracting a samplefrom the skin surface, and b.) measuring the glucose level from thesample.
 3. The process of claim 2, wherein the step of extracting isselected from the group consisting of blister suction, wick extraction,microdialysis extraction, iontophoretic extraction, sontophoreticextraction, and chemically enhanced extraction.
 4. The process of claim1, wherein the step of measuring comprises electrochemical sensors. 5.The process of claim 4, wherein the electrochemical sensors compriseglucose electrodes.
 6. The process of claim 1, wherein the step ofmeasuring comprises optochemical sensors.
 7. The process of claim 6,wherein the optochemical sensors comprise colorimetric strips.
 8. Theprocess of claim 1, wherein the step of measuring comprisesnear-infrared spectroscopy.
 9. The process of claim 1, wherein the stepof measuring comprises Raman spectroscopy.
 10. The process of claim 1,wherein the step of measuring comprises photoacoustic spectroscopy. 11.The process of claim 1, wherein the step of measuring comprisesmeasuring a refractive index or scatter changes of the sample.
 12. Theprocess of claim 1, wherein the step of measuring comprises fluorescentspectroscopy.
 13. The process of claim 1, wherein the step of measuringcomprises polarization spectroscopy.
 14. The process of claim 1 whereinthe period of time is less than six hours.
 15. The process of claim 1wherein the period of time is less than one hour.
 16. The process ofclaim 1 wherein the period of time is less than ten minutes.
 17. Theprocess of claim 1 wherein the period of time is less than five minutes.